Download 1X Equilibration/Wash Buffer (pH 7.0)

Protein Purification
https://www.youtube.com/watch?v=nY8jW9X
bYSY
Thrombin recognises the consensus sequence Leu-Val-Pro-Arg-Gly-Ser, cleaving the peptide bond between Arg
and Gly. This is utilised in many vector systems which encode such a protease cleavage site allowing removal of
an upstream domain. Predominantly the domain to be cleaved is a purification tag such as a 'His-Tag'
Column purification
Glutathion S-transferase
Purification of hexahistidine-tagged protein using Ni-chelated resin column
Lane OS: Original sample. Lane FT: Flowthrough. Lanes W1 and W2: Wash. Lane E1–E4:
Eluted 6xHN-AcGFP1 fractions
Protein purification
Materials
1X Equilibration/Wash Buffer (pH 8.0)
1X Imidazole Elution Buffer (pH 7.0)
Resin (TALON®, Clontech)
1M IPTG (Isopropyl β-D-1-thiogalactopyranoside)
1X Equilibration/Wash Buffer (pH 7.0)
50 mM sodium phosphate
8 M Urea
300 mM NaCl
1X Imidazole Elution Buffer (pH 7.0)
45 mM sodium phosphate
8 M Urea
270 mM NaCl
300 mM imidazole
Protein expression and purification from E. coli
1. Seed culture - Inoculate 5 ml of LB medium containing the appropriate antibiotics
(ampicillin) with a fresh bacterial colony harboring the expression plasmid. Grow at 37°C
2. Main culture - Dilute the non-induced overnight culture 1:100 (e.g., inoculate 25 ml
medium with 250 μl overnight culture) with fresh LB medium containing the appropriate
antibiotics (ampicillin). Grow at 37°C with vigorous shaking until the O.D 600 reaches
3. Add IPTG to a final concentration of 1 mM and grow the culture at 37°C with vigorous
shaking for 4~5 hours. (We grow the culture with shaking for 3 hours.)
4. Harvest the cells by centrifugation at 4000 rpm for 15 min at 4°C.
5. Resuspend the pellet in 2 ml of 1X Equilibration/Wash Buffer (pH 7.0) per 20–25 ml of
6. Gently agitate or stir the sample until it becomes translucent.
7. Centrifuge the sample at 10000 rpm for 20 min at 4°C to pellet any insoluble material.
8. Carefully transfer the supernatant to a clean tube without disturbing the pellet. This is the
Protein purification (TALON bead method)
1X Equilibration/Wash Buffer (pH 7.0)
Resin preparation
50 mM sodium phosphate
8 M Urea
300 mM NaCl
9. Thoroughly resuspend the TALON Resin. Immediately transfer 100 ul of resin suspension
10. Centrifuge at 1000 rpm for 2 min to pellet the resin. Remove and discard the supernatant.
11. Add 10 volumes (1 ml) of 1X Equilibration/Wash Buffer and mix briefly to pre12. Recentrifuge at 1000 rpm for 2 min to pellet the resin. Discard the supernatant.
Binding target protein to resin
13. Gently agitate at room temperature for 20 min on a platform shaker to allow the
polyhistidine-tagged protein to bind the resin.
14. Centrifuge at 1000 rpm for 5 min.
15. Carefully remove as much supernatant as possible without disturbing the resin pellet. (At
that time, we collet the supernatant. Do not discard supernatant.)
1X Equilibration/Wash Buffer (pH 7.0)
Washing
50 mM sodium phosphate
8 M Urea
300 mM NaCl
18. Wash the resin by adding 10 volumes (1 ml) of 1X Equilibration/Wash Buffer. Gently
agitate the suspension at room temperature for 10 min on a platform shaker
19. Centrifuge at 1000 rpm for 5 min. Remove and discard the supernatant. (At that time, we
collet the supernatant. Do not discard supernatant.)
20. Repeat Steps 18–19. (Do not forget to collect supernatant.)
Elution
1X Imidazole Elution Buffer (pH 7.0)
45 mM sodium phosphate
8 M Urea
270 mM NaCl
300 mM imidazole
21. Add 2 volumes (200 ul) of the 1X Imidazole Elution Buffer to the resin, and resuspend by
22. Centrifuge at 1000 rpm for 5 min. collect the supernatant.
23. Repeat Steps 21 and 22. (Do not forget to collect the supernatant.)
Protein denaturation: A process in which proteins lose the native structure
Proteins can be denatured by urea through several processes. One
method involves direct interaction whereby urea hydrogen bonds to
polarized areas of charge, such as peptide groups. This mutual
influence weakens the intermolecular bonds and interactions,
weakening the overall secondary and tertiary structure. Once
gradual protein unfolding occurs, water and urea can access more
easily the hydrophobic inner core of the protein in question,
speeding up the denaturation process
guanidine-HCl
Amino Acid Structure and molecular weight
Posttranslational modification
Hydrogen bond
Amino acids are linked
by H bonds
• Alpha helix
• Beta sheet
Folding of secondary structure
Hydrophobic/hydrophilic
Multi-subunit protein